Phenylalanine2-ornithine-vasopressin



Patented Feb. 27, 1968 United States Patent cc 3,371,080

1 2 r a 3 371 030 in which R denotes a radical capable of protecting aPHENYLALANINEZ 6RYETHINE8 VASOPRESSIN sulfhydryl radical in peptidesynthesis and R" denotes Roger Boissonnas Boflmingen, and Ren Huguem-n,a radical capable of protecting anamino radical in pep- Reinach,BaseLLand, Switzerland, assignol-s to Samioz tide synthesis, with areactive derivative of a free acid,

Ltd. (also known as Sandoz A.G.), Basel, Switzerland Saidfree acidhaving Formula 111 i No Drawing. Filed June 1, 1964, Ser. No. 371,762

S-R O H C H Claims priority, application Switzerland, June 6, 1963, 1 5a 5 7 123/63 CH2 CH2 CH: The portion of'the term of the patentsubsequent to J; 011

Jan. 17, 1984, has been disclaimed 0Y5 Phe Phe 1H 4 Claims. 01.zen-112.5 10 The present invention relates to a new polypeptide and inwhich R and R" have the above significance, to give to a process for theproduction thereof. the nonapeptide derivative of Formula IV,

. V HRII CONHz CH: R' $0 5 $0H5 (RH: ('10 NHa ?R C H2? H2 (IE9 (3H: CH2CH2 CIIHz (3H2 (3H2 (3H2 (3H2 R"NHCH-C ONHCHC O-NH-OH-C O-NH-CH-CO-NH-OH-C O-NH-CH-C O-NOHC 0-NHCHC ONH-CH2-C ONH; Cys Phe Phe Glu AspOys Pro Orn Gly IV The present invention provides the polypeptide ofForin which R and R" have the above significance, conmula I verting thisto the nonapeptide of Formula V, by splitv CONHz (EH2 (Elm (1 11 (3HN112 CHz-CH: l0H; CH; (llHz (13H! 3H2 CH2 CH5 N His-(EH43 O-NH-CHCO-NHCH-CONHCHCO-NHCHC ONH{OHCONH-CO-NHC HC ONHC Hz--C ON Hz r r s p sCys P be P he G111 A sp Cys P re Orn Gly I and its acid addition salts.ting off the protective radicals R and R", and converting The presentinvention also provides a process for the this to the Compound I byoxidation as above. production of a polypeptide of Formula I and itsacid Compounds II and the corresponding free hexapeptide addition salts,which comprises oxidizing the nonapeptide also form part of the presentinvention. of Formula V, It will be appreciated that it is Within thescope of the i T C ON H2 (llHa $11 C5115 C6115 $H2 CONH: SH CHz-(PH:(3H2 on, on, 0H, (3H1 3H2 (3H2 'risng (13H:NHz-CHCO-NH-CH-OONHGH-CONH-CH-CO-NHCH-CO- NHCHCON-CH-CONHCHCO-NHCHCONH2in aqueous solution at a pH value from about 4 to about presentinvention to produce Compound I starting with 9 and, when it is desiredto produce an acid addition Compound IV above, irrespective of themethod used for salt, reacting the resulting Compound I with an organicproducing Compound IV.

or inorganic acid. The nonapeptide derivative 1V may be obtained, forThe polypeptide V may be obtained by methods for example, bycondensation of two polypeptides other than the synthesis of compoundsof this type in actual use or the ones indicated above (or anoctapeptide and an amino described in the literature on the subject, itbeing possible acid) in the form of their protected derivatives. to jointogether the amino acids in the order indicated Examples of radicals forprotecting the sulfhydryl radiin the above Formula V one at a time or byfirst formcal in the above compounds are the phenyl, benzyl, ingconstituent peptide units and joining these together. p-bromobenzyl,p-chlorobenzyl, p-nitrobenzyl or p-Xylyl One method of producingCompound I and its acid radicals, While examples of radicals forprotecting the s addition salts comprises condensing a hexapeptidederivaamino radical are the carbobenzoxy, p-chlorobenzyloxytive ofFormula II, carbonyl, p-toluenesulfonyl or triphenylmethyl radicals.

bfi'HR/l (N112 211, 0H, oloNHi S|R' CHg-CH: (IJHz CH1 CH2 CH2 CH2 CH2Glu Asp Oys Pro 0 rn Gly II The starting materials for producing thenonapeptide derivative V by methods other than the one described hereinmay be obtained by methods for the synthesis of peptides in actual useor described in the literature on the subject, it being possible to jointogether the amino acids one at a time or by first forming constituentpeptide units and joining these together.

As indicated above, oxidation of the nonapeptide V to give the CompoundI is effected in accordance with the invention by oxidizing in aqueoussolution at a pH value of from about 4 to about 9. The oxidation ispreferably effected by the introduction of air, oxygen or hydrogenperoxide; an oxidizing agent, e.g., potassium ferricyanide, can likewisebe used.

The polypeptide I may be converted into its acid addition salts byreaction with an organic or inorganic acid. Examples of acids suitablefor acid salt formation with Compound I are as follows: hydrochloric,hydrobromic, sulfuric, fumaric, malic, maleic, acetic and tartaricacids.

structurally, Compound I resembles the two natural vasopressins thoughin each case two of the nine constituent amino acid residues aredifferent as may be seen from the following:

It is suggested that, when Compound I is used in an operation underlocal anaesthesia, it should be administered in admixture with the localanaesthetic, while when it is used in an operation under generalnarcosis, it should be administered in the form of a dilutephysiological sodium chloride solution. Compound I is further suggestedfor use orally in the therapeutical treatment of certain cases ofhypotonia or in the treatment of shocks and collapses.

Compound I may be used as free base or as theaddition salt with anorganic or inorganic acid, either on its own or in the form ofappropriate medicinal preparations for administration, e.g., orally,parenterally, enterally or intranasally. In order to produce suchmedicinal preparations, the Compound I and/or its acid addition salt isworked up with organic or inorganic adjuvants which are inert andphysiologically acceptable. Examples of such adjuvants are as follows:

Tablets and drages: lactose, starch, talc and stearic acid.

Syrups: solutions of cane sugar, invert sugar and glucose.

It will be seen that Compound I, i.e., Formula VI-c, (i) contains aphenylalanyl radical in the place of the tyrosyl radical present in bothvasopressin from pigs (Formula VI-a) and vasopressin from cattle(Formula VI-b) and (ii) contains an ornithine radical in the place ofthe lysine radical present in pig vasopressin or of the arginine radicalpresent in cattle vasopressin respectively.

Compound I has a vasoconstrictive action equal to that of the naturalvasopressins; however, in contradistinction to the natural vasopressins,the antidiuretic action of Compound I is, to a large extent, absent, andit is thus suggested for use in therapy as a substance having a specificvasoconstrictive effect. This specific vasoconstrictive effect ofCompound I results from a direct influence on the vascular muscles; forthis reason no appreciable side effects on the vegetative nervous systemare produced, as is the case with adrenalin and noradrenalin. Theproperties of Compound I are especially useful in the prophylaxis andtherapy of parenchymatous bleeding, whereby infiltration of the tissueswith Compound I produces a pronounced ischaemic effect. The propertiesof Compound I are, furthermore, of special use in surgery of the throat,nose and ear, in gynaecology and obstetrics, in urology and dentistry.

Injectable solutions: water, alcohols, glycerin and vegetable oils.

Suppositories: natural or hardened oils and waxes.

The preparations may furthermore contain suitable preserving,stabilizing or wetting agents, solubilizers, sweetening and coloringsubstances or flavorings.

The Compound I may be administered in approximately the followingdosages:

(a) In admixture with a local anaesthetic: 5 to 30 I.U./ 100 ml.solution.

(b) Infiltration of the operation area in the case of a generalanaesthetic: 10 to I.U./ ml. of'physiological saline solution.

(c) Instillation or application by means of pledget: 10 to 70 I.U./l00ml. of physiological saline solution.

(d) Systemic application in the case of circulatory collapse: 5 to 50I.U./50O ml. of physiological saline solution or 5% glucose solutionadministered by slow infusion.

Suitable ampules are, for example, those of 1 ml. containing 5 I.U. orthose of 10 ml. containing 15 I.U.

The present invention thus also provides pharmaceutical compositionscontaining, in addition to a physiologically acceptable carrier,Compound I and/or an acid addition salt thereof.

EXAMPLE 1 (a) N-a-carbobenzoxy-N-8-p-t0luene.s'u[f0nyl-L-ornilhyl-glycine ethyl ester 104 g. ofN-a-carbobenzoxy-N-fi-p-toluenesulfonyl-L- ornithine and 27 g. ofglycine ethyl ester are dissolved in 450 cc. of acetonitrile, themixture is cooled at 0, 51g. of dicyclohexylcarbodiirnide are added andthe mixture is shaken at room temperature for- 4 hours. Precipitateddicyclohexyl urea is filtered off and washed with acetonitrile. Thewhole filtrate is evaporated in a vacuum. The residue crystallizes afterthe addition of petroleum ether. After recrystallization fromn-propanol, 93 g. of N a carbobenzoxy N 6 p toluenesulfonyl L-ornithyl-glycine ethyl ester are obtained; melting point 135; [a] =7(96% ethanol).

(b) N-carbobenzovcy-L-prolyl-N-a-p-taluenesulfonyl-L-ornithyl-glycinamide 90 g. of N-a-carbobenzoxy-N-fi-p-toluenesulfonyl-L-ornithyl-glycine ethyl ester are dissolved in 800 cc. of anhydrousacetic acid which has been saturated with hydrogen bromide. The mixtureis left to stand for one hour at 20, evaporated in a vacuum at atemperature below 40 and the residue Washed carefully with diethylether. The residue is dissolved in 500 cc. of acetonitrile, 25 cc. oftriethylamine and 43 g. of N-carbobenzoxy-L- proline are added, coolingis effected at 0, 35.5 g. of dicyclohexylcarbodiimide are then added andthe mixture shaken overnight at 20". After filteringolf dicyclohexyl.

'urea, the filtrate is evaporated in a vacuum at 30,the

residue dissolved in ethyl acetate and this solution is washed withdilute sulfuric acid and aqueous ammonia.-

After drying over sodium sulfate, the ethyl acetate is removed byevaporation in a vacuum and the residue dissolved in 1 litre of absoluteethanol. The solution is cooled at 0, saturated with ammonia and left tostand overnight at 20 C. After evaporating in a vacuum at 30, theresidue is recrystallized from acetonitrile. 58 g. of N carbobenzoxy Lprolyl N 6 p toluenesulfonyl-L-ornithyl-glycinamide are obtained meltingpoint 122 (with decomposition); [a] =-46 (95% glacial acetic acid).

(c) N carbobenzoury L glutaminyl Lasparaginylsulfonyl-L-ornithyl-glycinamide 100 .g. ofN-carbobenzoxy-L-prolyl-N-fi-p-toluenesulfonyl-L-ornithyl-glycinamideare dissolved in 500 cc. of anhydrous acetic acid which has beensaturated with hydrogen bromide, the solution is left to stand for onehour at 20 and is evaporated in a vacuum at a tempera ture below 40 Theresidue is carefully washed with diethyl ether and then added to asolution of 100 g. of N carbobenzoxy L glutaminyl L asparaginyl S-benzyl-L-cysteinyl-azide and 26 cc. of triethylamine in 1000 cc. ofdimethylformamide. The mixture is left to stand overnight at 20, 3000cc. of ethyl acetate are added thereto, the precipitate is filtered offand washing is effected with ethyl acetate. 105 g. of N-carbobnzoxy-Lglutaminyl L asparaginyl S benzyl- L cysteinyl L- prolyl N 5 ptoluenesulfonyl L ornithyl glycinamide are obtained; melting point 193";[a] =-39 (dimethylformamide).

(d) N carbobenzoxy S-benzyI-L-cysteinyl-Lphenylwlanyl L phenylalanylL-glwtaminyl-L-asparagirzyl-S- benzyl Lcysteinyl-L-prolyl-N-6-pet0luenestrlfonyll washed with warm methanol. 45g. of

cut in-tubes 57 mide and the solution is left to stand for one hour at20". After evaporating the solvent in a vacuum at a term perature below40, the residue is carefully washed with diethyl ether and a solution of31.5 g. of N-carbobenzoxy- S benzylL-cysteinyl-L-phenylal-anyl-L-phenylalanineazide and 7.5 cc. oftrlethylamine in 250 cc. of dimethylformamide is added thereto. Themixture is left to stand for 2 days at 20, 1000 cc. of ethyl acetate aresubsequently added and the precipitate is washed with ethyl acetate.After drying in a vacuum at 30, the product is N-carbobenzoxy- S benzylL cysteinyl-L-phenyl-alanyl-L-phenylalanyl- L glutaminyl Lasparaginyl-S-benzyl-L-cysteinyl-L- prolyl N 6 p-toluen-esulfonylL-ornithyl-glycinamide are obtained; melting point 224?; [a] ==4-0(dimethylforrnamide) A l (e) L cysteinyl L phenylalanylL-phenylalany'l-L- glutaminyl L asparaginyl- L-cysteinyl-L-prDlyl-L-0rnithyl-glycinamide The necessary amount of sodium or potassium metalis added to a solution of 5 g. of N-carbobenzoxy-S-benzyl- L cysteinyl Lphenylalanyl L-phenylalanyl-L-glutaminyl L asparaginyl Sbenzyl-L-cysteinyl-L-prolyl- N 6 ptoluenesulfonyl-L-ornithyl-glycinamide in 1200 cc. of dry liquidammonia, while stirring at the boiling temperature of the solution, togive a stable blue coloration. After the addition of 3 g. of ammoniumchloride, the solution is evaporated to dryness. The residue contains Lcysteinyl L phenylalanyl-L-phenylalanyla L glutaminyl --'Lasparaginyl-L-cysteinyl-L-Pfolyl-L- ornithyl-glycinamide.

r Polypeptide Compound I 4.0-5.0 with dilute hydrochloric acid and afterthe addition of 50 g. of sodium chloride or 0.64 g. of methanesulfonicacid or 0.7 6 g. of trifluoroacetic acid, evaporation to dryness iseffected, whereby a dry powder results which keeps well. It may bestored and when used it may be dissolved to give a clear solution.However, the solution may also be used as such, if desiredafter'diluting with water or a salt solution.

H For purposes of removing the inorganic salts, the above owder,obtained after the addition of trifiuoroacetic acid, may be subjected tocounter-current distribution in the system secondarybutanol/Water/trifluoroacetic acid 1210116011. After 200 transfer stagesthe substance is presto 77 with a maximum in tube 67 (K=0.50). Afterevaporation, the active polypeptide is obtained with a good yield in theform of a hydroscopic tri'fluoroacetate, with uniform behavior inchromatography and electrophoresis. Migration in paper electrophoresisat a pH value of 5.8 and 40 V/cm.; 50 mm. in 60 minutes (the hlstidineused as reference migrates 65 mm). Migration in paper electrophoresis ata pH value of 1.9 and 40 V/cm.: 69 mm. in 60 minutes the tryptophaneused as reference migrates 66 mm). R in paper chromatography in thesystem isoamyl alcohol/pyridine/ water 35 :35p30: 0.28. Total hydrolysis(1 6 hours, HCl 6 N) yields the following amino acids in equimolecularquantities: cystine, glutaminic acid, asparaginic acid, proline,ornithine and glycine; and in double equirnolecular quantity:phenylalanine. The compound has the following biological activities:IU/rng. on the blood pressure of the rat, 15 IU/mg. on the inhibition ofdiuresis of the rat and less than 1 IU/mg. on the uterus of the rat.

of 7.5 cc. of a N solution of hydrogen peroxide in water 3,371,080 7 8EXAMPLE 2 and its pharmaceutically acceptable, non-toxic, acid additionsalts wherein all amino acid groupings except glycine are ofL-configuration.

3. A compound of the formula The same procedure as in Example 1 is used,except that final oxidation is eifected at 040 by the addition at a pHvalue of 7.58.0 (instead of oxidation by introducing air on oxygen).

wherein R is a peptide synthesis sulfhydryl radical pro- EXAMPLE 3tector selected from the group consisting of phenyl, The same procedureas in Example 1 is used, except 20 benzyl, p-brorno'benzyl,p-chlorobenzyl, p-nitrobenzyl that final oxidation is effected at 035 bythe addition of and p-xylyl, and I 6.7 cc. of a N solution of potassiumferricyanide in R is a peptide synthesis amino radical protector,wherewater at a pH value of 5.5-7.5. in all amino acid groupings exceptglycine are of Leon- What is claimed is: figuration.

1. A compound selected from the group consisting of a 25 polypeptide offormula:

NEH C|70NH2 (EH2 Ca s ([JcHs (EH2 CONHz CID-CH1: $Hz (EH: (iZHI CI-IqCIIz (I /Hz CIHZNHi-orp-oo-NH-oH-o0-NHoH-oo-NH-dHooNHciH-co-NHoH-ooNoH-oo-NH-oHCONHOH:-C o NH2 $11: (1112 S S Cys Phe Phe Glu Asp Oys Pro Orn Gly Iand is pharmaceutically acceptable, non-toxic, acid addi- 40 tion saltswherein all amino acid groupings except glycine 4. The compoundN-carbobenzoxy-S-benzyl-L-cysteinare of L-configuration. yl Lphenylalanyl L phenylalanyl L glutaminyl- 2. A compound selected fromthe group consisting of a L asparaginyl S benzyl L cysteinyl L prolyl N-nonapeptide of formula: 5 p toluenesulfonyl L ornithyl glycinamide.

T' CONE: (3H1 H (10115 (75115 (ll Hz CIONHz H CHz-CFH: (3H2 (I311, (3H1CH: (I311; (1H (I111, 0H2 (3H1NHa-CHCONHCHCO-NHH-CO-NHCHCONHGHCONHCHCON-CHOO-NHC HC ONHCH2 C O NH:

References Cited UNITED STATES PATENTS 3,299,036 1/1967 Boissonnas etal. 260--112.5

OTHER REFERENCES Boissonnas et al., Experientia 17, 377-390 (1961).

LEWIS GOTTS, Primary Examiner.

JULIAN S. LEVI'IT, ELBERT L. ROBERTS,

Examiners.

M. M. KASSENOFF, S. J. FRIEDMAN,

Assistant Examiners.

1. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF A POLYPEPTIDE OFFORMULA: